Since the first gene targeting experiments in yeast more than 25 years ago (Hinnen et al, 1978; Rothstein, 1983), homologous recombination (HR) has been used to insert, replace or delete genomic sequences in a variety of cells (Thomas and Capecchi, 1987; Capecchi, 2001; Smithies, 2001). HR is a very conserved DNA maintenance pathway involved in the repair of DNA double-strand breaks (DSBs) and other DNA lesions (Paques and Haber, 1999; Sung and Klein, 2006), but it also underlies many biological phenomenon, such as the meiotic reassortiment of alleles in meiosis (Roeder, 1997). A competing pathway in DSBs repair events is the Non-Homologous End Joining (NHEJ) pathway which accounts for all DSBs repair events in the absence of an homologous repair matrix (Paques and Haber, 1999; van Gent et al, 2001). Although perfect relegation of the broken ends is probably the most frequent event, imperfect rejoining of the broken ends can result in the addition or deletion of one of several base pairs, inactivating the targeted open reading frame. Homologous gene targeting strategies have been used to knock out endogenous genes (Capecchi, M. R., Science, 1989, 244, 1288-1292, Smithies, O., Nature Medicine, 2001, 7, 1083-1086) or knock-in exogenous sequences in the chromosome. It can as well be used for gene correction, and in principle, for the correction of mutations linked with monogenic diseases. However, this application is in fact difficult, due to the low efficiency of the process (10−6 to 10−9 of transfected cells). The frequency of HR can be significantly increased by a specific DNA double-strand break (DSB) at a locus (Rouet et al, 1994; Choulika et al, 1995). Such DSBs can be induced by meganucleases, sequence-specific endonucleases that recognize large DNA recognition target sites (12 to 30 bp).
Meganucleases show high specificity to their DNA target, these proteins being able to cleave a unique chromosomal sequence and therefore do not affect global genome integrity. Natural meganucleases are essentially represented by homing endonucleases, a widespread class of proteins found in eukaryotes, bacteria and archae (Chevalier and Stoddard, 2001). Early studies of the I-Scel and HO homing endonucleases have illustrated how the cleavage activity of these proteins can be used to initiate HR events in living cells and have demonstrated the recombinogenic properties of chromosomal DSBs (Dujon et al, 1986; Haber, 1995). Since then, meganuclease-induced HR has been successfully used for genome engineering purposes in bacteria (Posfai et al, 1999), mammalian cells (Sargent et al, 1997; Donoho et al, 1998; Cohen-Tannoudji et al, 1998), mice (Gouble et al, 2006) and plants (Puchta et al, 1996; Siebert and Puchta, 2002).
Other specialized enzymes like integrases, recombinases, transposases and endonucleases have been proposed for site-specific genome modifications. For years, the use of these enzymes remained limited, due to the challenge of retargeting their natural specificities towards desired target sites. Indeed, the target sites of these proteins, or sequences with a sufficient degree of sequence identity, should be present in the sequences neighboring the mutations to be corrected, or within the gene to be inactivated, which is usually not the case, except in the case of pre-engineered sequences.
Meganucleases have emerged as scaffolds of choice for deriving genome engineering tools cutting a desired target sequence (Paques et al. Curr Gen Ther. 2007 7:49-66). Combinatorial assembly processes allowing to engineer meganucleases with modified specificities has been described by Arnould et al. J Mol. Biol. 2006 355:443-458; Arnould et al. J Mol. Biol. 2007 371:49-65; Smith et al. NAR 2006 34:e149; Grizot et al. NAR 2009 37:5405. Briefly, these processes rely on the identifications of locally engineered variants with a substrate specificity that differs from the substrate specificity of the wild-type meganuclease by only a few nucleotides.
Although these powerful tools are available, there is still a need to further modulate double-strand break-induced homologous recombination and more particularly to increase the efficiency of gene targeting, i.e. the frequency of integration events of an exogenous gene at a targeted locus.
RNA interference is an endogenous gene silencing pathway that responds to dsRNAs by silencing homologous genes (Meister, G. & Tuschl, T., 2004). First described in Caenorhabditis elegans by Fire et al, the RNAi pathway functions in a broad range of eukaryotic organisms (Hannon, G. J. et al, 2002). Silencing in these initial experiments was triggered by introduction of long dsRNA. The enzyme Dicer cleaves these long dsRNAs into short-interfering RNAs (siRNAs) of approximately 21-23 nucleotides. One of the two siRNA strands is then incorporated into an RNA-induced silencing complex (RISC). RISC compares these “guide RNAs” to RNAs in the cell and efficiently cleaves target RNAs containing sequences that are perfectly, or nearly perfectly complementary to the guide RNA.
For many years it was unclear whether the RNAi pathway was functional in cultured mammalian cells and in whole mammals. However, Elbashir S. M. et al, 2001, triggered RNAi in cultured mammalian cells by transfecting them with 21 nucleotide synthetic RNA duplexes that mimicked endogenous siRNAs. McCaffrey et al, 2002, also demonstrated that siRNAs and shRNAs could efficiently silence genes in adult mice.
Introduction of chemically synthetized siRNAs can effectively mediate post-transcriptional gene silencing in mammalian cells without inducing interferon responses.
Synthetic siRNAs, targeted against a variety of genes, have been successfully used in mammalian cells to prevent expression of target mRNA (Harborth J. et al, 2001).
These discoveries of RNAi and siRNA-mediated gene silencing has led to a spectrum of opportunities for functional genomics, target validation, and the development of siRNA-based therapeutics, making it a potentially powerful tool for therapeutics and in vivo studies.
It has been demonstrated that inhibition of genes implicated in NHEJ stimulates HR and gene targeting (Allen et al, 2002; Delacote et al, 2002; Bertolini et al, 2009). NHEJ inhibition has been achieved either by using mutants, either by inhibition of gene expression through siRNAs.
In WO2007/013979, the expression of six genes supposed to be implied in NHEJ, Ku70, Ku86, DNA-PKcs, XRCC4, DNA ligase IV and Artemis, are silenced to show that these genes are clearly decreasing the random integration of a linearized GFP vector and are slightly increasing targeted integration of a HPRT matrix-like at the HPRT locus.
WO2008/113847 relates to a bipartite gene-replacement method, resulting in a combined recombination and targeted integration event in a parent eukaryotic cell with a preference for Non homologous Recombination (NHR), said eukaryotic cell having an increased HR/NHR ratio by deleting hdfA or hdfB gene of Penicillium chrysogenum, respectively fungal equivalents of Ku70 and Ku80 Saccharomyces cerivisiae genes.
None of these techniques allowed identifying genes implicated in double-strand break-induced HR.
Slabicki et al. briefly summarizes a method aiming at identifying genes involved in double strand break repair. This method is based on the measure of gene conversion events, and not of gene targeting events. This document fails to provide an accurate and detailed description of the method. In addition, the method only led to the identification of very few genes. Moreover, this document neither teaches nor suggests that modulating the identified gene in a eukaryotic cell could be useful for increasing targeted integration of a transgene.
It is thus highly desirable to construct new cell lines in which double-strand break-induced HR can be modulated, particularly in which genome targeting of a polynucleotide or gene of interest can take place with higher frequency.
Methods, agents and compositions that could be used to modulate double-strand break-induced HR would be extremely advantageous, particularly to increase the integration efficiency of a transgene into a genome at a predetermined location.